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Brain mitochondrial DNA is not damaged by prolonged cardiac arrest or reperfusion
B C White1, R C Tribhuwan, D J Vander Laan
1Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, Michigan 48201.
Abstract:
Postischemic reperfusion is known to cause iron-mediated peroxidation of polyunsaturated fatty acids in membranes, including mitochondrial membranes, in the brain cortex. Consequently, we tested the hypothesis that this radical-mediated damage would extend to DNA. Mitochondrial DNA (mtDNA) was chosen because of its presence at a known site of free radical formation, its sensitivity and ease of assay, and its known lack of any repair systems. In model experiments we utilized endonuclease III or piperidine to amplify topological form conversions in mtDNA damaged by in vitro reactions with hydroxyl radical. We then applied the amplified detection assays to dog brain mtDNA isolated after 2 or 8 h of reperfusion following a 20-min cardiac arrest. We found that ischemia and reperfusion caused no topological form conversions in mtDNA. Similarly, nucleotide incorporation by a gap-filling reaction showed no sensitivity to digestion of the mtDNA by exonuclease III, an enzyme known to remove blocked 3' termini at the site of radical-generated nicks. Furthermore, the recovery of mtDNA was similar in all experimental groups, suggesting that putatively damaged forms had not been removed by rapid degradation. Thus, despite mitochondrial membrane damage, brain mtDNA does not accumulate oxygen radical damage during postischemic brain reperfusion.
Insights
Postischemic reperfusion causes membrane damage but not DNA damage in brain mitochondria. Despite mitochondrial membrane injury, brain mitochondrial DNA (mtDNA) remains protected from oxygen radical damage during reperfusion.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Postischemic reperfusion can cause iron-mediated peroxidation of polyunsaturated fatty acids in brain mitochondrial membranes.
- This process generates free radicals, raising concerns about potential damage to mitochondrial DNA (mtDNA).
Purpose of the Study:
- To test the hypothesis that radical-mediated damage during postischemic reperfusion extends to brain mitochondrial DNA (mtDNA).
- To investigate the integrity of mtDNA following ischemia and reperfusion in the brain cortex.
Main Methods:
- Utilized endonuclease III or piperidine in model experiments to amplify topological form conversions in hydroxyl radical-damaged mtDNA.
- Applied amplified detection assays to dog brain mtDNA isolated after 2 or 8 hours of reperfusion following cardiac arrest.
- Assessed mtDNA integrity using nucleotide incorporation assays with exonuclease III and monitored mtDNA recovery rates.
Main Results:
- Ischemia and reperfusion did not cause topological form conversions in brain mtDNA.
- Nucleotide incorporation assays showed no sensitivity to exonuclease III digestion, indicating no radical-generated nicks.
- mtDNA recovery rates were similar across experimental groups, suggesting no rapid degradation of damaged forms.
Conclusions:
- Brain mitochondrial DNA (mtDNA) does not accumulate oxygen radical damage during postischemic reperfusion.
- Despite observed mitochondrial membrane damage, mtDNA remains protected from oxidative insults in the brain cortex.